Pathology (February 2006) 38(1), pp. 58–62
EXPERIMENTAL PATHOLOGY
Lithium-induced lung toxicity in rats: the effect of caffeic acid phenethyl ester (CAPE) ONDER SAHIN*, OSMAN SULAK{, YUCEL YAVUZ{, EFKAN UZ§, IBRAHIM ERENd, H. RAMAZAN YILMAZ§, MEHMET ALI MALAS{, IRFAN ALTUNTAS" AND AHMET SONGUR** Afyon Kocatepe University, School of Medicine, Departments of *Pathology, {Emergency Medicine and **Anatomy, Afyon, Turkey; Suleyman Demirel University, School of Medicine, Departments of {Anatomy, §Medical Biology, dPsychiatry and "Biochemistry, Isparta, Turkey
Summary Aims: We aimed to evaluate the effects of caffeic acid phenethyl ester (CAPE) on lithium (Li)-induced lung toxicity. Methods: Twenty-two adult male Wistar albino rats weighing between 280 and 300 g were used. The rats were randomly divided into three groups: control, Li and Li+CAPE groups. Li and CAPE were co-administered intraperitoneally twice daily for 4 weeks. Control rats were given 0.9% NaCl during the same period. All the rats were allowed to feed ad libitum until midnight after they had received the proposed treatment. Results: In the Li group, peribronchial and intraparenchymal lymphocyte and macrophage infiltration were observed. Atypical type II pneumocytes, alveolar destruction and emphysematous changes were also detected. Lymphocyte and macrophage infiltration was significantly decreased in the Li+CAPE group compared with the Li group. Alveolar destruction, emphysematous changes and intraparenchymal mononuclear cell infiltration were also recovered to a level close to the control group. Malondialdehyde (MDA) levels were increased in the Li group compared with the control group. CAPE administration decreased the MDA levels in the Li+CAPE group. Conclusions: CAPE was found to associate with histopathological changes recovery in the lungs and oxidative stress due to Li treatment. Key words: Lithium, CAPE, lung, toxicity, histopathology, MDA. Received 19 July, revised 5 October, accepted 12 October 2005
INTRODUCTION Lithium carbonate is a very invaluable and widely used drug for the treatment of several psychiatric disorders.1,2 However, prolonged treatment with therapeutic levels of lithium (Li) causes toxic side effects.3 Besides this, permanent neurological, cardiac, hepatic and renal damage have been reported as a result of Li intoxication.4–7 It has been suggested that oxidative stress, one of the principal toxic effects of Li, results in increased lipid peroxidation in the cerebral cortex and kidney.8 Oral Li intake has been reported to cause toxic effects in several organs such as the testis,3 heart,4 brain7 and kidney.5,8 These studies indicate the possibility that the respiratory system might also be affected by Li via the
systemic circulation. Indeed, Greenspan et al.9 found that acute exposure to Li aerosol (2600, 2300, 1400, or 620 mg/ m3 for 4 h) caused suppurative bronchopneumonia or aspiration pneumonia in F344/Lov rats. However, Johansson et al.10 found that Li chloride inhalation (0.6 and 1.9 mg/m3 for 6 h/day, 5 days/week for 4–8 weeks) had no significant toxic effects in lungs of rabbits. In our previous report we have shown that 25 mg/kg of Li carbonate twice daily for 4 weeks caused renal tubular damage and oxidative stress in rats and that this can be prevented by caffeic acid phenethyl ester (CAPE).11 CAPE is one of the major components of honeybee propolis extracts and has been used as a folk medicine for many years.12,13 CAPE has several biological and pharmacological properties such as anticarcinogenic,14 antiviral15 and immunomodulatory16 activities. Also, recently, CAPE has been reported to be a new potent antioxidant and antiinflammatory agent.17–20 CAPE treatment was reported to protect the spinal cord from ischaemia-reperfusion injury,21 the kidney from ischaemia-reperfusion injury22 and the testis from torsion and detorsion damages.11 The present study aimed to assess the potential toxic effects of Li treatment and possible protective effects of CAPE on rats’ lung tissue morphology and biochemistry.
MATERIALS AND METHODS Animals and treatment Twenty-two adult male Wistar albino rats (mean weight between 280 and 300 g) obtained from the Laboratory Animal Production Unit of Suleyman Demirel University, Turkey, were used in the experiment. This study was approved by Suleyman Demirel University Animal Ethical Committee and the rats were maintained and used in accordance with the ‘Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Suleyman Demirel University, Animal Ethical Committee’. The rats were randomly divided into three groups, as follows: control group (n58), lithium-treated group (Li, n57), and lithium + CAPE treated group (Li+CAPE, n57). Li was administered intraperitoneally with 25 mg/kg Li2CO3 solution in 0.9% NaCl twice daily for 4 weeks as defined previously.11,23 CAPE (Sigma Chemical Co., USA) was co-administered intraperitoneally at a dose of 10 mM/kg/day once a day for 4 weeks.11 Control rats were treated with 0.9% NaCl during the same period. All of the rats were allowed to feed ad libitum until midnight after they had received the proposed treatment. Rats were anaesthetised with an intramuscular injection of 50 mg/kg ketamine hydrochloride (Ketalar; Eczacibasi, Turkey) and killed 24 h after the last injection.
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia DOI: 10.1080/00313020500464904
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The lungs were removed, and each divided into two for biochemical analyses and histopathological study. Tissue samples for biochemical study were washed with physiological saline and suspended in 3 mL pH 7.3 TrisHCl buffer that contained 0.25 M sucrose. Rat lungs were stored at 280uC until biochemical analysis. For histopathological study, other tissue samples were fixed in 10% neutral buffered formalin. Tissue homogenate For biochemical analyses, the lungs of the rats were washed with physiological saline. They were then homogenised for 3 min (UltraTurrax T25; IKA Werke, Germany) in cold phosphate buffer in order to provide a 10% homogenate. These homogenates were centrifuged at 6000 g for 10 min to obtain supernatants. The levels of protein and malondialdehyde (MDA) were determined in the supernatants. Protein content of homogenates was determined by the Lowry method.24 Biochemical analysis MDA levels, an indicator of free radical generation which increases at the end of the lipid peroxidation, were estimated by the double heating method of Hadley and Draper.25 The principle of the method was spectrophotometric measurement of the color developed during reaction to thiobarbituric acid (TBA) with MDA. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 mL homogenate in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling under tap water, the mixture was centrifuged at 1000 g for 10 min, and 2 mL of the supernatant was added to 1 mL of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled under tap water and its absorbance was measured using a Shimadzu UV1601 spectrophotometer (Shimadzu, Japan) at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA-TBA complex 1.566105/cm/M and expressed in nmol/mg of protein. Histological examination After dehydration procedures, the samples were blocked in paraffin; 4–6mm sections were cut with a microtome and stained with H&E. Mounted slides were examined and photographed under a light microscope (Nikon THP117; Nikon, Japan). In the criteria for evaluations of the peribronchial inflammation, intraparenchymal infiltration, atypical type II pneumocytes, nodular aggregates, macrophages in alveolar lumen, alveolar destruction and emphysematous changes were based on intensity and diffusion of staining in the samples. Intensity and diffusion of these observations were separately numbered from 0 to 3+ semiquantitatively. Following this procedure, the counted values were summed in each section, recombining the values of intensity and diffusion (called the ‘degree of staining’) as follows: 0, no staining; 1+, minimal; 2+, low; 3+, moderate; 4+, strong; 5+, heavy; and 6+, most heavy.26,27 Statistical analysis All data were presented as mean¡standard deviation (SD). Statistical analysis was performed on a personal computer using SPSS for Windows software (SPSS Inc., USA). The data of biochemical and histopathological changes were considered to be non-parametric, therefore they were performed using the Kruskal–Wallis and Mann–Whitney U tests. p,0.05 was considered statistically significant.
RESULTS All the rats survived throughout the experimental period. Blood lithium levels were found to be 0 in the control group, whereas they were between 0.5 and 0.8 mmol/L in Li and Li+CAPE groups. The level of MDA increased significantly in the Li group compared with the control group and decreased significantly in the Li+CAPE group compared with the Li group (p,0.05; Fig. 1).
Fig. 1 MDA levels in rats’ lungs between the groups. Bars represent mean¡SD. a,b Different superscripts show significant differences between groups (p,0.05; Mann–Whitney U test).
The histopathological findings are summarised in Table 1 and Fig. 2. Under light microscopic examination, lungs of control rats showed a regular histological structure (Fig. 2A). In the Li group, however, peribronchial and intraparenchymal mononuclear cell (lymphocyte, macrophage) infiltration was observed. Intraparenchymal lymphocyte infiltration was diffuse and follicle formation was detected. In addition, atypia in type II pneumocytes, alveolar destruction and emphysematous changes were also observed (Fig. 2B–E). However, co-administration of CAPE resulted in a significantly decreased peribronchial and intraparenchymal lymphocyte and macrophage infiltration when compared with the Li group rats. Alveolar destruction, emphysematous changes and intraparenchymal mononuclear cell infiltration were also prevented to a level close to that of the control group (Fig. 2F).
DISCUSSION Li is frequently used as an effective drug for the treatment or prophylaxis of several psychiatric disorders such as bipolar affective disorder in humans.28 It is known to cause biochemical changes and morphological alterations in the liver, kidney, heart, brain and testes at higher doses or after prolonged therapeutic use.3,5,28–31 Reviewing the literature, no information was obtained about biochemical and morphological changes in the lungs at therapeutic doses of oral Li carbonate and the effects of CAPE on these changes. One of the aims of the present study was to investigate the probable toxic effects of Li on rats’ lungs. We also aimed to determine whether CAPE prevented this toxicity. A subtoxic dose of lithium carbonate (25 mg/kg body weight) was applied intraperitoneally in our study. We have previously reported that at 25 mg/kg Li caused renal tubular damage and oxidative stress in rats, which was abrogated by CAPE.11 Therefore, we hypothesised that at a similar dosage lung tissues may be adversely affected by Li and that this could be prevented by CAPE. In addition, the toxic effects of Li on other organs such as the testis,3 heart4 and brain7 indicate that Li might affect the respiratory
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TABLE 1 Comparison of the histopathological changes in the control (I), lithium (II) and Li+CAPE (III) groups Peribronchial infiltration
Intraparenchymal infiltration
Atypical type II pneumocytes
Nodular aggregates
Macrophages in alveolar lumen
Alveolar destruction
Emphysematous changes
1.91 4.00 2.38
1.52 3.89 1.63
0.00 2.11 1.50
0.72 3.78 0.50
0.29 2.11 1.25
0.00 2.11 0.25
0.00 2.11 0.25
0.022
0.031
0.010
0.002
0.013
0.001
0.001
I–III
NS
NS
II–III
NS
NS 0.009
NS 0.002
NS 0.002
Group I II III P values I–II
0.037 NS
NS 0.001
NS
NS, non-specific (p.0.05).
Fig. 2 Histopathological findings in rats’ lungs among the three groups. (A) Normal histological appearance in control rat (H&E, 640). (B) Peribronchial and intraparenchymal infiltrations, alveolar destruction and emphysematous changes in Li group rat (H&E, 640). (C) Diffuse and nodular aggregates and alveolar destruction in Li group rat (H&E, 6100). (D,E) Intraparenchymal infiltrations, alveolar destruction and emphysematous changes as well as atypical type II pneumocytes and alveolar macrophages (histiocytes) in Li group rat (H&E, 6200). (F) Histological appearance in Li+CAPE group rat (H&E, 640). , peribronchial infiltration; , intraparenchymal infiltration; R, alveolar destruction; *, emphysematous change; P, atypical type II pneumocyte; H, alveolar macrophage (histiocyte).
CAPE PREVENTS LITHIUM-INDUCED LUNG TOXICITY
system via the systemic circulation. In our study, the increase of MDA level in the lungs of the Li-administered rats suggests that oxidative stress like lipid peroxidation might have formed. The observation of inflammation in the histopathological investigations supports this argument. It is already known that oxidative stress might lead to inflammation. CAPE, an active propolis component, has antioxidant, antimicrobial, anti-inflammatory, carcinostatic and immunomodulatory properties. CAPE was also shown to inhibit lipo-oxygenase activity and thereby suppress the lipid peroxidation. Nuclear transcription factor kappa B (NFkB) inhibition is involved in most of these activities. CAPE has been shown to block specifically and completely the activation of NF-kB induced by a wide variety of inflammatory agents including tumour necrosis factor (TNF) and H2O2.21 According to our study results, we speculate that CAPE may inhibit the activation of NF-kB. CAPE treatment was reported to protect the lung from bleomycine-related lung fibrosis,20 ischaemia-reperfusion injury,32 lung cancer33 and other lung injuries.34 In our study, morphological and biochemical changes in lung tissue due to Li carbonate and the effects of CAPE on these changes were evaluated. Histopathologically, peribronchial and intraparenchymal mononuclear cell infiltration, atypical type II pneumocytes, alveolar destruction and emphysematous changes were observed in the lungs of rats treated with Li. These changes were considered as secondary to inflammation. It was also found that the levels of MDA, a marker of lipid peroxidation in lung tissue, were increased in the same rats. This suggests that oxidative stress such as lipid peroxidation may have occurred. In the rats co-administered with Li and CAPE, peribronchial and intraparenchymal lymphocyte and macrophage infiltration, atypical type II pneumocytes, alveolar destruction and emphysematous changes were statistically significantly decreased. In addition, a decrease of MDA levels and inflammation in the Li+CAPE group rats was observed, attesting the role of CAPE as a strong antioxidant. Johansson et al.10 administered aerosolised Li chloride (0.6 and 1.9 mg/m3, 6 h/day, 5 days/week for 4–8 weeks) to rabbits and examined the lungs under light and electron microscopes. They reported no significant effects of Li inhalation on the rabbit lungs. However, in order to investigate the effects of Li use in fusion reactors on human health, Greenspan et al.9 studied rats that inhaled inflammable aerosols of Li (2600, 2300, 1400, or 620 mg/m3 for 4 h) and evaluated the histopathological findings. They found ulcerative or necrotic laryngitis, focal or segmental rhinitis or squamous cell metaplasia, supurative bronchopneumonia and aspiration pneumonitis in the respiratory system. The findings of this study indicate that Li, at the high doses, has acute injurious effects on the lungs. Biochemically, MDA, a marker of lipid peroxidation in the lung tissue, was found to be statistically higher in Li group rats than the control group (p,0.05). Previous studies reported increased lipid peroxidation in brain cortex and kidney due to lithium.8,11 Several studies have been conducted to reveal the causality of free radicals in the pathogenesis of a great variety of toxic substances.11,20,22,33,35 In conclusion, histopathological and biochemical changes in the lung tissue due to Li toxicity were observed
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in our study. We showed that CAPE markedly prevented Li-induced inflammatory injury and oxidative stress in the lungs. However, detailed studies such as myeloperoxidase and Na+-K+ ATPase activity would further our knowledge on the mechanism of Li toxicity in the lung. If future reports support our findings, CAPE could be useful for preventing lung injury in severe psychiatric disorders requiring long term Li use. Address for correspondence: Dr O. Sahin, Izmir Istasyon Cad. Mecidiye Mah. Avcı Apt. Kat:4 No:9, Afyon, Turkey. E-mail:
[email protected]
References 1. Xu J, Culman J, Blume A, et al. Chronic treatment with a low dose of lithium protects the brain against ischemic injury by reducing apoptotic death. Stroke 2003; 34: 1287–92. 2. Gupta AK, Knowles SR, Gupta MA, et al. Lithium therapy associated with hidradenitis suppurativa: case report and a review of the dermatologic side effects of lithium. J Am Acad Dermatol 1995; 32: 382–6. 3. Thakur SC, Thakur SS, Chaube SK, et al. Subchronic supplementation of lithium carbonate induces reproductive system toxicity in male rat. Reprod Toxicol 2003; 17: 683–90. 4. Frassati D, Tabib A, Lachaux B, et al. Hidden cardiac lesions and psychotropic drugs as a possible cause of sudden death in psychiatric patients: a report of 14 cases and review of the literature. Can J Psychiatry 2004; 49: 100–5. 5. Chmielnicka J, Nasiadek M. The trace elements in response to lithium intoxication in renal failure. Ecotoxicol Environ Saf 2003; 55: 178–83. 6. Johnson GF. Lithium in depression: a review of the antidepressant and prophylactic effects of lithium. Aust NZ J Psychiatry 1987; 21: 356–65. 7. Simard M, Gumbiner B, Lee A, et al. Lithium carbonate intoxication. A case report and review of the literature. Arch Intern Med 1989; 149: 36–46. 8. Sawas AH, Gilbert JC. Lipid peroxidation as a possible mechanism for the neurotoxic and nephrotoxic effects of a combination of lithium carbonate and haloperidol. Arch Int Pharmacodyn Ther 1985; 276: 301–12. 9. Greenspan BJ, Allen MD, Rebar AH. Inhalation toxicity of lithium combustion aerosols in rats. J Toxicol Environ Health 1986; 18: 627–37. 10. Johansson A, Camner P, Curstedt T, et al. Rabbit lung after inhalation of lithium chloride. J Appl Toxicol 1988; 8: 373–5. 11. Oktem F, Ozguner F, Sulak O, et al. Lithium-induced renal toxicity in rats: Protection by a novel antioxidant caffeic acid phenethyl ester. Mol Cell Biochem 2005; 277: 109–15. 12. Uz E, Sogut S, Sahin S, et al. The protective role of caffeic acid phenethyl ester (CAPE) on testicular tissue after testicular torsion and detorsion. World J Urol 2002; 20: 264–70. 13. Yilmaz HR, Iraz M, Sogut S, et al. The effects of erdosteine on the activities of some metabolic enzymes during cisplatin-induced nephrotoxicity in rats. Pharmacol Res 2004; 50: 287–90. 14. Chen YJ, Shiao MS, Wang SY. The antioxidant caffeic acid phenethyl ester induces apoptosis associated with selective scavenging of hydrogen peroxide in human leukemic HL-60 cells. Anticancer Drugs 2001; 12: 143–9. 15. Fesen MR, Kohn KW, Leteurtre F, et al. Inhibitors of human immunodeficiency virus integrase. Proc Natl Acad Sci USA 1993; 90: 2399–403. 16. Song YS, Park EH, Jung KJ, et al. Inhibition of angiogenesis by propolis. Arch Pharm Res 2002; 25: 500–4. 17. Rao CV, Desai D, Simi B, et al. Inhibitory effect of caffeic acid esters on azoxymethane-induced biochemical changes and aberrant crypt foci formation in rat colon. Cancer Res 1993; 53: 4182–8. 18. Sud’ina GF, Mirzoeva OK, Pushkareva MA, et al. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993; 329: 21–4. 19. Michaluart P, Masferrer JL, Carothers AM, et al. Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Res 1999; 59: 2347–52. 20. Ozyurt H, Sogut S, Yildirim Z, et al. Inhibitory effect of caffeic acid phenethyl ester on bleomycine-induced lung fibrosis in rats. Clin Chim Acta 2004; 339: 65–75. 21. Ilhan A, Koltuksuz U, Ozen S, et al. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/reperfusion injury in rabbits. Eur J Cardiothorac Surg 1999; 16: 458–63.
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22. Gurel A, Armutcu F, Sahin S, et al. Protective role of alpha-tocopherol and caffeic acid phenethyl ester on ischemia-reperfusion injury via nitric oxide and myeloperoxidase in rat kidneys. Clin Chim Acta 2004; 339: 33–41. 23. Leschiner S, Weizman R, Shoukrun R, et al. Tissue-specific regulation of the peripheral benzodiazepine receptor by antidepressants and lithium. Neuropsychobiology 2000; 42: 127–34. 24. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75. 25. Hadley M, Draper HH. Identification of N-(2-propenal) serine as a urinary metabolite of malondialdehyde. FASEB J 1988; 2: 138–40. 26. Songur A, Akpolat N, Kus I, et al. The effects of the inhaled formaldehyde during the early postnatal period in the hippocampus of rats: A morphological and immunohistochemical study. Neurosci Res Commun 2003; 33: 168–78. 27. Fidan F, Unlu M, Sezer M, et al. Acute effects of environmental tobacco smoke and dried dung smoke on lung histopathology in rabbits. Pathology 2006; 38: 53–57. 28. Shaldubina A, Agam G, Belmaker RH. The mechanism of lithium action: state of the art, ten years later. Prog Neuropsychopharmacol Biol Psychiatry 2001; 25: 855–66.
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29. Chatterjee S, Roden K, Banerji TK. Morphological changes in some endocrine organs in rats following chronic lithium treatment. Anat Anz 1990; 170: 31–7. 30. Rodriguez-Gil JE, Fernandez-Novell JM, Barbera A, et al. Lithium’s effects on rat liver glucose metabolism in vivo. Arch Biochem Biophys 2000; 375: 377–84. 31. Tandon A, Dhawan DK, Nagpaul JP. Effect of lithium on hepatic lipid peroxidation and antioxidative enzymes under different dietary protein regimens. J Appl Toxicol 1998; 18: 187–90. 32. Calikoglu M, Tamer L, Sucu N, et al. The effects of caffeic acid phenethyl ester on tissue damage in lung after hind limb ischemiareperfusion. Pharmacol Res 2003; 48: 397–403. 33. Chen MF, Wu CT, Chen YJ, et al. Cell killing and radiosensitization by caffeic acid phenethyl ester (CAPE) in lung cancer cells. J Radiat Res 2004; 45: 253–60. 34. Koksel O, Kaplan MB, Ozdulger A, et al. Oleic acid-induced lung injury in rats and effects of caffeic acid phenethyl ester. Exp Lung Res 2005; 31: 483–96. 35. Ozen S, Akyol O, Iraz M, et al. Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats. J Appl Toxicol 2004; 24: 27–31.
